The project goal of this U01 application is to develop a combination of synthetic immunostimulatory agents that maximizes host resistance to potentially lethal microbial agents. Most pathogens important for biodefense are first encountered at mucosal or epithelial sites in the respiratory tree or the gastrointestinal tract. Many microbial agents display ligands that activate one or more Toll-like receptors (TLRs). Dendritic cells (DCs) at mucosal sites play a crucial role in immune responses against infections by sensing microbial invasion through TLRs. However, DC activation by microbial agents may be compromised at the mucosal surfaces of normal people, because of a tolerogenic immune environment. Based upon our preliminary experiments, we believe that this local immune deficit can be overcome by delivering to mucosal surfaces an optimized combination of TLR ligands, together with a potentiator of TLR signaling. Our proposed experiments will focus on activators of TLR7 and TLR9, which are coexpressed by DCs. In some cases, the TLR ligands will be combined with synthetic inhibitors of IMP dehydrogenase (IMPDH), which have been shown to potentiate TLR signaling by inducing interferon regulatory factors. The proposed experiments also will exploit several cell and animal model systems that should predict clinical activity. This project has three development milestones: 1. To compare novel synthetic activators of TLR7 and 9, with a cell nonspecific activator of TLR2, for their abilities to induce cytokine and co-stimulatory molecule synthesis by murine and human cells, used alone and in combination with IMPDH inhibitors. 2. To analyze the effects of the synthetic TLR ligands and IMPDH inhibitors on innate and adaptive immunity in animals after mucosal and systemic administration. 3. To test the efficacies of the optimized drug combinations to protect mice from lethal bacterial and viral infections. When complete, these experiments will deliver a rationally conceived and thoroughly evaluated product for clinical testing.